Factor VII (FVII) is a zymogen for a vitamin K‐dependent serine protease essential for the initiation of blood coagulation. It is synthesized primarily in the liver and circulates in plasma at a concentration of approximately 0.5 μg/ml (10 nmol/L). The FVII gene (F7) is located on chromosome 13 (13q34), consists of 9 exons, and spans approximately 12kb. It encodes a mature protein of 406 amino acids, which has an N‐terminal domain (Gla) post‐translationally modified by γ‐carboxylation of glutamic acid residues, two domains with homology to epidermal growth factor (EGF1 and 2), and a C‐terminal serine protease domain. The single chain zymogen is activated by proteolytic cleavage at Arg152‐Ile153. There are 238 individuals described in the world literature with mutations in their F7 genes (FVII mutation database; europium.csc.mrc.ac.uk). Complete absence of FVII activity in plasma is usually incompatible with life, and individuals die shortly after birth due to severe hemorrhage. The majority of individuals with mutations in their F7 gene(s), however, are either asymptomatic or the clinical phenotype is unknown. In general, a severe bleeding phenotype is only observed in individuals homozygous for a mutation in their F7 genes with FVII activities (FVII:C) below 2% of normal, however, a considerable proportion of individuals with a mild‐moderate bleeding phenotype have similar FVII:C by in vitro assay. The failure of in vitro tests to differentiate between these groups may be due to lack of sensitivity in the assays to the very low amounts of FVII:C, which are sufficient to initiate coagulation in vivo. A number of polymorphisms have been identified in the F7 gene and some have been shown to influence plasma FVII antigen levels. Hum Mutat 17:3–17, 2001. © 2001 Wiley‐Liss, Inc.

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5′ splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor–like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the ▵EGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans. © 1998 by The American Society of Hematology.

Epidermal growth factor‐like (EGF) domains are one of the more abundant modules found in functionally diverse extracellular proteins. The domain is characterized by six cysteine residues, which normally disulphide bond in a 1–3, 2–4, 5–6 pattern. A particular subset (cbEGF) contains the consensus sequence [DEQN]‐x‐[DEQN] 2 ‐x m ‐[DN]*‐x n ‐[YF] (where m and n are variables and * indicates possible β‐hydroxylation) associated with the ability to bind calcium. At present, cbEGFs have three main functional roles, as a spacer unit, in protein–protein interactions, and in structural stabilization. High‐resolution structures of cbEGF domains have identified a common fold, comprising a major and a minor double‐stranded β‐hairpin, stabilized by the three consensus disulphide bonds. A single calcium ion binds to the amino terminus and is coordinated by a pentagonal bipyramidal arrangement of oxygen atoms. Each individual cbEGF domain contains a calcium‐binding site, whose affinity may be considerably enhanced by N‐terminal linkage to another domain. A number of inherited diseases caused by missense mutations within cbEGFs highlight the importance of the domain.

DNA single-strand breaks (SSBs) arising as a result of the disruption of phosphodiester bond linkage of nucleotides in a polymer are dangerous, because, if left unrepaired, they may block vital processes such as DNA transcription and DNA replication. SSBs arise by several distinct mechanisms including direct energy deposition by ionizing radiation, attack by reactive oxygen species, during enzymatic processing of endogenous DNA lesions, and as a result of aberrant DNA topoisomerase I activity (reviewed in [1,2]). Many of the SSBs arise as a consequence of loss of the DNA base and subsequent sugar fragmentation, which should be considered as a single nucleotide gap with modified 5¢ and ⁄ or 3¢ ends. For example, SSBs produced by ionizing radiation or by attack from reactive oxygen species often contain 3¢-phosphoglycolate or 3¢-phosphate groups [3]. Similarly, some radiationinduced SSBs, as well as those arising during base excision repair (BER), contain a 5¢-sugar phosphate residue [4][5][6]. Thus, SSBs produced by several genotoxic agents include a variety of termini that have to be converted into conventional 3¢-OH ⁄ 5¢-phosphate nicks before the gap can be filled by a DNA polymerase and the DNA ends resealed by a DNA ligase. Several BER enzymes have been shown to possess 'end cleaning' activities. The major mammalian endonuclease that processes abasic sites [apurinic ⁄ apyrimidinic endonuclease 1 (APE1)] [7] was also shown to be the major 3¢-phosphoglycolate activity in human cell extracts [8][9][10][11]. Human polynucleotide kinase (PNK) is the major 3¢-phosphatase [12,13], and DNA polymerase b (Pol b) is the major activity in human cell extracts catalysing removal of 5¢-sugar phosphate residues [14]. Although most of the enzymes involved in end processing have been identified and this process plays a key role in maintaining genome stability, the precise mechanism governing recognition and processing of such a variety of SSBs is unclear. To Ionizing radiation, oxidative stress and endogenous DNA-damage processing can result in a variety of single-strand breaks with modified 5¢ and ⁄ or 3¢ ends. These are thought to be one of the most persistent forms of DNA damage and may threaten cell survival. This study addresses the mechanism involved in recognition and processing of DNA strand breaks containing modified 3¢ ends. Using a DNA-protein cross-linking assay, we followed the proteins involved in the repair of oligonucleotide duplexes containing strand breaks with a phosphate or phosphoglycolate group at the 3¢ end. We found that, in human whole cell extracts, end-damage-specific proteins (apurinic ⁄ apyrimidinic endonuclease 1 and polynucleotide kinase in the case of 3¢ ends containing phosphoglycolate and phosphate, respectively) which recognize and process 3¢-end-modified DNA strand breaks are required for efficient recruitment of X-ray cross-complementing protein 1-DNA ligase IIIa heterodimer to the sites of DNA repair.Abbreviations SSB, single-strand break; BER, base excision repair; APE1, apurinic ⁄ apyri...

The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterolrich lipoproteins from the circulation. Mutations to the receptor result in familial hypercholesterolemia, and over 60 of these occur in the calcium-binding epidermal growth factor-like domain pair. Two selected mutations in this region (G322S and R329P) were introduced into the domain pair and analyzed by in vitro refolding. Both exhibited differing levels of protein misfolding with R329P being the most pronounced. Solution NMR studies of the mutant domain pairs after purification established that a fraction of protein maintains a native-like fold and that this fraction contains two intact calciumbinding sites. An in vivo analysis of intact receptors containing these binding sites showed significantly reduced cell-surface expression compared with the native LDL receptor levels, again with R329P showing the most severe decrease. The sum of these results suggests that either local changes in structure or domain misfolding may be associated with the mutations. There is also the possibility that the misfolding of the calcium-binding epidermal growth factor-like pair region is propagated to other regions of the intact receptor, resulting in more global defects. Surprisingly, for both mutants, those fulllength receptors that fold and reach the cell surface retain the ability to bind LDL and release the ligand upon exposure to low pH. This analysis provides significant insight into the protein defect resulting from each of the two mutations and allows their classification to be 2B (partially transport-defective). The results also highlight a range of misfolding defects that may be associated with familial hypercholesterolemia and may enable the prediction of the consequences of homologous disease-causing mutations to other proteins. The low density lipoprotein (LDL)1 receptor mediates the cellular uptake of cholesterol-rich lipoproteins from the circulation by binding to two ligand molecules, apolipoprotein B and apolipoprotein E, of LDL and ␤-very LDL particles. Receptorligand complexes enter the cell through endocytosis at clathrincoated pits. The bound lipoprotein is released subsequently in the low pH environment of the endosome, and the receptors are recycled back to the cell surface (1).Mutations to the LDL receptor (LDLR) are associated with familial hypercholesterolemia (FH), an autosomal-dominant inherited disease affecting ϳ1 in 500 in most populations. FH is characterized clinically by a lifelong elevation of LDL-bound cholesterol in blood and is associated with an increased risk of coronary artery disease and cardiovascular disease (2). Almost 900 LDLR mutations have been associated with FH (3), collated in two online databases, 2 and many have been classified based on their effect on the synthesis (class 1), transport (class 2), ligand binding activity (class 3), internalization (class 4), or recycling (class 5) of the LDLR (4). Despite the wealth of biochemical data available for the LDL receptor and...

Plain English summaryUsher syndrome is the most common cause of deafblindness worldwide and is estimated to affect between 3 and 6 people in every 100,000. Children are born with hearing loss and develop sight loss in their early years of life. A barrier to the involvement and participation of deafblind people in research is access to information in appropriate formats. The degree of sight and hearing impairment experienced by individuals is variable, so there is not a one size fits all solution. We held a research discussion group, that included five people with Usher syndrome, to consider people’s accessibility needs for an upcoming research project involving this condition.We have identified a number of considerations for including deafblind people in conversations about research: i) using appropriately sized meeting rooms which offer control over lighting, layout and sound; ii) where appropriate, ensuring written/printed materials are high contrast (e.g. black text with a yellow background) and in large (18 point and above), sans-serif fonts (e.g. Arial); iii) identifying the relevant communication support for the individual whether that be sign language interpretation, lip reading, hearing loop, speech to text reporting or a combination; iv) ensuring that there is access to emotional support for both people who are deafblind and their families before, during and after the research.The outcome of this work is a checklist of considerations when planning to hold a research conversation with someone who is deafblind and hinges on earlier interactions to identify the appropriate support needs for the individual.Abstract Background Usher syndrome is the most common cause of deafblindness worldwide. Children are born with hearing loss and develop sight loss in their early years of life. It is estimated to affect between 3 and 6 people in every 100,000. A barrier to the involvement and participation of deafblind people in research is access to information in appropriate formats. Individuals have varying degrees of sight and hearing impairment meaning there is not a singular solution to supporting all people’s communication needs. There is evidence that severe sight and hearing impairments are used as exclusion criteria in some research studies. This exclusion may extend into involvement activities. Methods Eight people, including five people with Usher syndrome, attended a research discussion group. Through this activity, we identified what to consider when looking to improve the experience of taking part in a discussion about research for deafblind individuals. Results Among contributors two people made use of standard British Sign Language interpretation and one communicated using hands-on signing. Contributors highlighted the limitations associated with signing and lip reading such as exhaustion and clear lines of sight as well as the need for additional formats such as speech to text reporting, and high contrast (e.g. black text with a yellow background) printouts with large (18 point and above), sans-serif font...

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We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5′ splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor–like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the ▵EGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans. © 1998 by The American Society of Hematology.